Electrochemical aptasensors for pathogenic detection toward point-of-care diagnostics.

A biosensor system refers to a biomedical device, which detects biological, chemical, or biochemical components by converting those signals to an electrical signal by utilizing and uniting physical or chemical transducer with biorecognition elements. An electrochemical biosensor is generally based on the reaction of either production or consumption of electrons under a three-electrode system. Biosensor systems are exploited in a wide range of areas, such as medicine, agriculture, husbandry, food, industry, environment protection, quality control, waste disposal, and the military. Pathogenic infections are the third leading cause of death worldwide after cardiovascular diseases and cancer. Therefore, there is an urgent need for effective diagnostic tools to control food, water, and soil contamination result in protecting human life and health. Aptamers are peptide or oligonucleotide-based molecules that show very high affinity to their targets that are produced from large pools of random amino acid or oligonucleotide sequences. Generally, aptamers have been utilized for fundamental sciences and clinical implementations for their target-specific affinity and have been intensely exploited for different kinds of biosensor applications for approximately 30 years. The convergence of aptamers with biosensor systems enabled the construction of voltammetric, amperometric, and impedimetric biosensors for the detection of specific pathogens. In this review, electrochemical aptamer biosensors were evaluated by discussing the definition, types, and production techniques of aptamers, the advantages of aptamers as a biological recognition element against their alternatives, and a wide range of aptasensor examples from literature in the detection of specific pathogens.

Detection of Microorganisms Using Graphene-Based Nanobiosensors.

Having an insight into graphene and graphene derivatives such as graphene oxide, reduced graphene oxide and graphene quantum dots is necessary since it can help scientists to detect possible properties and features that could be useful when using these carbon materials in preparation of a nanocomposites. In recent years, graphene and its derivatives have attracted a lot of attention and been extensively applied in biosensors due to fascinating properties, such as large surface area, optical and magnetic properties, and high elasticity for the detection of microorganisms as they can be modified with some other materials such as macromolecules, oxide metals and metals to improve the electrochemical behaviour of the biosensor. In this review paper, biosensor design strategies based on graphene and its derivatives (graphene-based nanocomposites in biosensors) are described. Then their application for the detection of microorganisms including prions, viroids, viral and bacterial cells as well as fungi, protozoa, microbial toxins and even microbial sources of antibiotics is reviewed.

Fluorogenic 7-azidocoumarin and 3/4-azidophthalimide derivatives as indicators of reductase activity in microorganisms.

A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.

Fluorogenic l-alanylaminopeptidase substrates derived from 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins and their potential applications in diagnostic microbiology.

Six novel fluorogenic enzyme substrates for detecting l-alanylaminopeptidase activity in microorganisms have been prepared and evaluated in Columbia agar media. The substrates are l-alanyl derivatives of 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins. Both the quinoline and coumarin series of substrates produced fluorescence in the presence of Gram-negative microorganisms. In contrast, fluorescence generation in the presence of the Gram-positive microorganisms and yeasts was limited or absent.

2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the detection of nitroreductase activity in clinically important microorganisms.

A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential nitroreductase substrates for the purpose of detecting clinically important microorganisms. Several of the substrates produced highly fluorescent colonies with the majority of a panel of 10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.

Signal amplification strategy of DNA self-assembled biosensor and typical applications in pathogenic microorganism detection.

Biosensors have emerged as ideal analytical devices for various bio-applications owing to their low cost, convenience, and portability, which offer great potential for improving global healthcare. DNA self-assembly techniques have been enriched with the development of innovative amplification strategies, such as dispersion-to-localization of catalytic hairpin assembly, and dumbbell hybridization chain reaction, which hold great significance for building biosensors capable of realizing sensitive, rapid and multiplexed detection of pathogenic microorganisms. Here, focusing primarily on the signal amplification strategies based on DNA self-assembly, we concisely summarized the strengths and weaknesses of diverse isothermal nucleic acid amplification techniques. Subsequently, both single-layer and cascade amplification strategies based on traditional catalytic hairpin assembly and hybridization chain reaction were critically explored. Furthermore, a comprehensive overview of the recent advances in DNA self-assembled biosensors for the detection of pathogenic microorganisms is presented to summarize methods for biorecognition and signal amplification. Finally, a brief discussion is provided about the current challenges and future directions of DNA self-assembled biosensors.

Potential Application of the WST-8-mPMS Assay for Rapid Viable Microorganism Detection.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process-the conventional standard for bacterial detection-is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection.

Ultrasensitive qPCR platform for rapid detection of bacterial contamination of raw biological samples at the point of care.

Contamination of cell cultures can result in a significant loss of precious biological material, particularly in long-term processes including amplification of chimeric antigen receptors (CAR)-T cells and differentiation of patient-derived stem cells, for therapeutic purposes. Bacterial contamination can also lead to more complex conditions such as sepsis which can cause morbidity and mortality, despite strict controls and good laboratory/manufacturing practices in the manipulation of complex biological samples such as blood used in autologous and allogeneic stem cells transplantation. The current standard method to identify biological risk is the set-up of microbial cultures, which can be time consuming with the likelihood of wasting large amounts of reagents in the event of contamination. Real-Time Polymerase Chain Reaction (qPCR) is a molecular method able to detect biological agents in a highly sensitive and specific way and in a short time. However, qPCR assays require complex DNA/RNA purification steps and expensive benchtop instruments, which may not always be available. This paper reports an extraction-free and low-volume protocol for qPCR in a standard instrument, which has been demonstrated to be effective on both Gram-positive (Gram+) and Gram-negative (Gram-) bacteria. Detection has been obtained from spiked cell culture samples, reaching a limit of detection (LOD) of 1 colony forming unit (CFU)/ml. To demonstrate the high potential of this optimized procedure, the same samples were also tested on a Point-Of-Care platform, which includes a cartridge with micro-chambers and a compact instrument, capable of performing qPCR with the same efficiency. Staphylococcus aureus (Gram+) was selected as the target for a proof of concept, achieving a LOD of 1 CFU/ml also on the portable device. The availability of these results paves the way for a simplified protocol for DNA extraction and amplification.

Aetiology of Community-Acquired Pneumonia and the Role of Genetic Host Factors in Hospitalized Patients in Cyprus.

Community-acquired pneumonia (CAP) remains the leading cause of hospitalization among infectious disease in Europe, and a major cause of morbidity and mortality. In order to determine and characterize the aetiology of CAP in hospitalized adults in Cyprus, respiratory and blood samples were obtained from hospitalized patients with CAP, and analyzed using Multiplex Real-Time PCR/RT-PCR, and ID/AMR enrichment panel (RPIP) analysis. Probe-based allelic discrimination was used to investigate genetic host factors in patients. The aetiology could be established in 87% of patients. The most prevalent viral pathogens detected were influenza A, SARS-CoV-2, and human rhinovirus. The most common bacterial pathogens detected were Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae. Antimicrobial resistance genes were identified in 23 patients. S. aureus was the most common AMR correlated strain in our study. A positive correlation was detected between bacterial infections and the NOS3 rs1799983 G allele and the FCGR2A rs1801274 G allele. A positive correlation was also detected between the TNF-α rs1800629 A allele and sepsis, while a negative correlation was detected with the ACE rs1799752 insertion genotype and the severity of pneumonia. In conclusion, the targeted NGS panel approach applied provides highly sensitive, comprehensive pathogen detection, in combination with antimicrobial resistance AMR insights that can guide treatment choices. In addition, several host factors have been identified that impact the disease progression and outcome.

Surface Functionalization and Escherichia coli Detection Using Surface-Enhanced Raman Spectroscopy Driven by Functional Organic Polymer/Gold Nanofilm-Based Microfluidic Chip.

In this work, a microfluidic prototype based on polymeric materials was developed to monitor surface processes using surface-enhanced Raman spectroscopy (SERS), keeping the reagents free of environmental contamination. The prototype was fabricated on poly(methyl methacrylic acid) (PMMA). A micrometric membrane of a functional organic polymer (FOP) based on p-terphenyl and bromopyruvic acid monomers was formed on the PMMA surface to promote the formation of metal nanoclusters. Au nanosized film was deposited on the FOP membrane to give rise to the SERS effect. A microchannel was formed on another piece of PMMA using micromachining. A representative 3D model of the prototype layer arrangement was built and simulated in COMSOL Multiphysics® to approximate the electric field distribution and calculate the power enhancement factor as the Au film changes over time. The fabrication process was characterized using UV-visible and Raman spectroscopies and XPS. The prototype was tested using a Raman microscope and liquid solutions of cysteamine and Escherichia coli (E. coli). The simulation results demonstrated that the morphological characteristics of the Au layer give rise to the SERS effect, and the power enhancement factor reaches values as high as 8.8 × 105 on the FOP surface. The characterization results showed the formation of the FOP and the Au film on PMMA and the surface functionalization with amine groups. The Raman spectra of the prototype showed temporal evolution as different compounds were deposited on the upper wall of the microchannel. Characteristic peaks associated with these compounds were detected with continuous monitoring over time. This prototype offers many benefits for applications like monitoring biological processes. Some advantages include timely surface evaluation while avoiding environmental harm, decreased use of reagents and samples, minimal interference with the process by measuring, and detecting microorganisms in just 1 h, as demonstrated with the E. coli sample.

Microfluidics for Environmental Applications.

Microfluidic and lab-on-a-chip systems have become increasingly important tools across many research fields in recent years. As a result of their small size and precise flow control, as well as their ability to enable in situ process visualization, microfluidic systems are increasingly finding applications in environmental science and engineering. Broadly speaking, their main present applications within these fields include use as sensors for water contaminant analysis (e.g., heavy metals and organic pollutants), as tools for microorganism detection (e.g., virus and bacteria), and as platforms for the investigation of environment-related problems (e.g., bacteria electron transfer and biofilm formation). This chapter aims to review the applications of microfluidics in environmental science and engineering - with a particular focus on the foregoing topics. The advantages and limitations of microfluidics when compared to traditional methods are also surveyed, and several perspectives on the future of research and development into microfluidics for environmental applications are offered.

The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection.

Fluorescent in situ hybridization (FISH) is a powerful tool that for more than 30 years has allowed to detect and quantify microorganisms as well as to study their spatial distribution in three-dimensional structured environments such as biofilms. Throughout these years, FISH has been improved in order to face some of its earlier limitations and to adapt to new research objectives. One of these improvements is related to the emergence of Nucleic Acid Mimics (NAMs), which are now employed as alternatives to the DNA and RNA probes that have been classically used in FISH. NAMs such as peptide and locked nucleic acids (PNA and LNA) have provided enhanced sensitivity and specificity to the FISH technique, as well as higher flexibility in terms of applications. In this review, we aim to cover the state-of-the-art of the different NAMs and explore their possible applications in FISH, providing a general overview of the technique advancement in the last decades.

Shotgun Proteomics for Food Microorganism Detection.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

Methodology for preparing a cosmetic sample for the development of Microorganism Detection System (SDM) software and artificial intelligence learning to recognize specific microbial species.

INTRODUCTION AND OBJECTIVE: The article presents the methodology of preparing a cosmetic sample for analysi, and the creation of a dataset for teaching artificial intelligence to recognize specific species of microorganisms in cosmetic samples in terms of compliance with the ISO standard document, to develop of the Microorganism Detection System (SDM). MATERIAL AND METHODS: Methodology of preparation a cosmetic sample for testing covers the steps from taking a cosmetic sample to obtaining separated living microorganisms through staining to photos, which in the final stage are used for analysis of the purity of cosmetics by SDM, as well as for learning and testing of the deep convolutional neural network (CNN) for detecting and classifying cells of specific species of bacteria, fungi and yeast in cosmetics, according to the document of standard PN-EN ISO 17516-2014:11. RESULTS: A new techique was devised for preparing a cosmetic sample for the development of Microorganism Detection System (SDM) software, and artificial intelligence learning to recognize specific microbial species. Based on metod demonstrated, the Intelligent algorithms of SDM proved to be effective in counting and recognizing specific microorganisms (average accuracy for Candida albicans - 97%, Escherichia coli - 76%, Pseudomonas aeruginosa - 70%, Staphylococcus aureus - 85%), which are the most important species for the assessment of the purity of cosmetics. In addition, the reproducibility of the developed method was verified, and the results obtained were comparable to the breeding methods currently used, based on specific standards. CONCLUSIONS: The experiments confirmed the high sensitivity and specificity of the SDM method, its repeatability and, above all, the comparability of the results with clasic methods of European standards.

Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection.

Pathogenic bacteria cause serious harm to human health, which calls for the development of advanced detection methods. Herein, we developed a novel detection platform based on fluorescence resonance energy transfer (FRET) for rapid, ultrasensitive and specific bacteria detection, where gold nanoparticles (AuNPs, acceptor) were conjugated with aptamers while upconversion nanoparticles (UCNPs, donor) were functionalized with corresponding complementary DNA (cDNA). The spectral overlap between UCNPs fluorescence emission and AuNPs absorption enables the occurrence of FRET when hybridizing the targeted aptamer and cDNA, causing upconversion fluorescence quenching. In the presence of target bacteria, the aptamers preferentially bind to bacteria forming a three-dimensional structure and thereby dissociate UCNPs-cDNA from AuNPs-aptamers, resulting in the recovery of upconversion fluorescence. Using the UCNPs based FRET aptasensor, we successfully detected Escherichia coli ATCC 8739 (as a model analyte) with a detection range of 5-106cfu/mL and detection limit of 3cfu/mL. The aptasensor was further used to detect E. coli in real food and water samples (e.g., tap/pond water, milk) within 20min. The novel UCNPs based FRET aptasensor could be used to detect a broad range of targets from whole cells to metal ions by using different aptamer sequences, holding great potential in environmental monitoring, medical diagnostics and food safety analysis.