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

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

Impedimetric Sensors for Bacteria Detection

The application of electrochemical biosensors based on impedance detection has grown during the past years due to their high sensitivity and rapid response, making this technique extremely useful to detect biological interactions with biosensor platforms. This chapter is focused on the use of electrochemical impedance spectroscopy (EIS) for bacterial detection in two ways. On one hand, bacteria presence may be determined by the detection of metabolites produced by bacterial growth involving the media conductivity changes. On the other hand, faster and more selective bacterial detection may be achieved by the immobilization of bacteria on a sensor surface using biorecognition elements (antibodies, antimicrobial peptides, aptamers, etc.) and registering changes produced in the charge transfer resistance (faradic process) or interfacial impedance (nonfaradic process). Here we discuss different types of impedimetric biosensors for microbiological applications, making stress on their most important parameters, such as detection limits, detection times, selectivity, and sensitivity. The aim of the paper was to give a critical review of recent publications in the field and mark the future trends

A Bifunctional Nanocomposites Based Electrochemical Biosensor for In-field Detection of Pathogenic Bacteria in Food

This research focused on the application of electrochemical biosensors for the rapid detection of pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium, in foods. The possible presence of pathogenic bacteria in foods has always been a great threat to the wellbeing of people and the revenue of food companies. Therefore, the demand for rapid and sensitive methods to detect foodborne pathogens is growing. In this research, an impedimetric immunosensor was first developed for the rapid detection of E. coli O157:H7 and S. Typhimurium in foods. It was based on the techniques of immunomagnetic separation, enzyme labelling, and electrochemical impedance spectroscopy (EIS). This impedimetric immunosensor was capable of specifically detecting E. coli O157:H7 and S. Typhimurium within the range of 102 to 106 colony forming unit (cfu)/ml in the pure culture. The limits of detection (LODs) of E. coli O157:H7 in ground beef and S. Typhimurium in chicken carcass rinse water were 2.05 x 103 cfu/g and 1.04 x 103 cfu/ml, respectively. The second electrochemical biosensor was designed for rapid detection of E. coli O157:H7. This biosensor integrated magnetic GOx-polydopamine (PDA) based polymeric nanocomposites (PMNCs) which served dual functions as both the carrier and the label, and Prussian blue (PB) modified SP-IDMEs for measurement. The core-shell Abs/GOxext/gold nanoparticles (AuNPs)/magnetic beads (MBs)-GOx@PDA PMNCs acted efficiently to get a high load of enzyme onto the surface of bacterial cells. A filtration step separated the free PMNCs from the bonded ones and reduce the background noise to achieve better sensitivity. The constructed biosensor had been proved to be able to detect E. coli O157:H7 with the LOD of 52 cfu/ml in the pure culture. The third electrochemical aptasensor was developed to detect S. Typhimurium based on the concept of the bifunctional nanocomposites. The ssDNA aptamers were used as the biorecognition element. The achieved LOD in the pure culture was 96 cfu/ml. The biosensors developed in this research exhibited good specificity, reproducibility, and easy-to-operate, and are expected to find broad applications in the detection, especially in-field detection, of foodborne pathogens

Recent Advancements in the Technologies Detecting Food Spoiling Agents

To match the current life-style, there is a huge demand and market for the processed food whose manufacturing requires multiple steps. The mounting demand increases the pressure on the producers and the regulatory bodies to provide sensitive, facile, and cost-effective methods to safeguard consumers’ health. In the multistep process of food processing, there are several chances that the food-spoiling microbes or contaminants could enter the supply chain. In this contest, there is a dire necessity to comprehend, implement, and monitor the levels of contaminants by utilizing various available methods, such as single-cell droplet microfluidic system, DNA biosensor, nanobiosensor, smartphone-based biosensor, aptasensor, and DNA microarray-based methods. The current review focuses on the advancements in these methods for the detection of food-borne contaminants and pathogens

Advanced biosensors for detection of pathogens related to livestock and poultry

Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review

Antimicrobial peptides: Powerful biorecognition elements to detect bacteria in biosensing technologies

Bacterial infections represent a serious threat in modern medicine. In particular, biofilm treatment in clinical settings is challenging, as biofilms are very resistant to conventional antibiotic therapy and may spread infecting other tissues. To address this problem, biosensing technologies are emerging as a powerful solution to detect and identify bacterial pathogens at the very early stages of the infection, thus allowing rapid and effective treatments before biofilms are formed. Biosensors typically consist of two main parts, a biorecognition moiety that interacts with the target (i.e., bacteria) and a platform that transduces such interaction into a measurable signal. This review will focus on the development of impedimetric biosensors using antimicrobial peptides (AMPs) as biorecognition elements. AMPs belong to the innate immune system of living organisms and are very effective in interacting with bacterial membranes. They offer unique advantages compared to other classical bioreceptor molecules such as enzymes or antibodies. Moreover, impedance-based sensors allow the development of label-free, rapid, sensitive, specific and cost-effective sensing platforms. In summary, AMPs and impedimetric transducers combine excellent properties to produce robust biosensors for the early detection of bacterial infectionsPeer ReviewedPostprint (published version

Emerging (Bio)Sensing Technology for Assessing and Monitoring Freshwater Contamination - Methods and Applications

Ecological Water Quality - Water Treatment and ReuseWater is life and its preservation is not only a moral obligation but also a legal requirement. By 2030, global demands will exceed more than 40 % the existing resources and more than a third of the world's population will have to deal with water shortages (European Environmental Agency [EEA], 2010). Climate change effects on water resources will not help. Efforts are being made throughout Europe towards a reduced and efficient water use and prevention of any further deterioration of the quality of water (Eurostat, European Comission [EC], 2010). The Water Framework Directive (EC, 2000) lays down provisions for monitoring, assessing and classifying water quality. Supporting this, the Drinking Water sets standards for 48 microbiological and chemical parameters that must be monitored and tested regularly (EC, 1998). The Bathing Water Directive also sets concentration limits for microbiological pollutants in inland and coastal bathing waters (EC, 2006), addressing risks from algae and cyanobacteria contamination and faecal contamination, requiring immediate action, including the provision of information to the public, to prevent exposure. With these directives, among others, the European Union [EU] expects to offer its citizens, by 2015, fresh and coastal waters of good quality

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification