Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2.

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104-7.0 × 107 CFU mL-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

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-Coll@MNPs) for bacteria expressing collagen binding adhesins was 1.3 times higher than that of arginine MNPs (Arg@MNPs), herein used as control. More importantly, after optimization of the MNP concentration and contact time, the H-Coll@MNPs were able to capture 95% of bacteria present in the samples. More importantly, the bacteria can be enriched within 30 min and the time for bacterial identification is effectively shortened in comparison to the "gold standard" in clinical diagnosis. These results suggest that H-Coll@MNPs can be used for the selective isolation of specific bacteria from mixed populations present, for example, in biological samples.

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.

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.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

New solutions to capture and enrich bacteria from complex samples.

Current solutions to diagnose bacterial infections though reliable are often time-consuming, laborious and need a specific laboratory setting. There is an unmet need for bedside accurate diagnosis of infectious diseases with a short turnaround time. Moreover, low-cost diagnostics will greatly benefit regions with poor resources. Immunoassays and molecular techniques have been used to develop highly sensitive diagnosis solutions but retaining many of the abovementioned limitations. The detection of bacteria in a biological sample can be enhanced by a previous step of capture and enrichment. This will ease the following process enabling a more sensitive detection and increasing the possibility of a conclusive identification in the downstream diagnosis. This review explores the latest developments regarding the initial steps of capture and enrichment of bacteria from complex samples with the ultimate goal of designing low cost and reliable diagnostics for bacterial infections. Some solutions use specific ligands tethered to magnetic constructs for separation under magnetic fields, microfluidic platforms and engineered nano-patterned surfaces to trap bacteria. Bulk acoustics, advection and nano-filters comprise some of the most innovative solutions for bacteria enrichment.

Exploring the potential of polyethylene terephthalate in the design of antibacterial surfaces.

Polyethylene terephthalate (PET) is one of the most used polymeric materials in the health care sector mainly due to its advantages that include biocompatibility, high uniformity, mechanical strength and resistance against chemicals and/or abrasion. However, avoiding bacterial contamination on PET is still an unsolved challenge and two main strategies are being explored to overcome this drawback: the anti-adhesive and biocidal modification of PET surface. While bacterial adhesion depends on several surface properties namely surface charge and energy, hydrophilicity and surface roughness, a biocidal effect can be obtained by antimicrobial compounds attached to the surface to inhibit the growth of bacteria (bacteriostatic) or kill bacteria (bactericidal). Therefore, it is well known that granting antibacterial properties to PET surface would be beneficial in the prevention of infectious diseases. Different modification methods have been reported for such purpose. This review addresses some of the strategies that have been attempted to prevent or reduce the bacterial contamination on PET surfaces, including functionalisation, grafting, topographical surface modification and coating. Those strategies, particularly the grafting method seems to be very promising for healthcare applications to prevent infectious diseases and the emergence of bacteria resistance.