Matrix Effects on the Microcystin-LR Fluorescent Immunoassay Based on Optical Biosensor

Matrix effects on the microcystin-LR fluorescent immunoassay based on the evanescent wave all-fiber immunosensor (EWAI) and their elimination methods were studied. The results indicated that PBS and humic acid did not affect the monitoring of samples under the investigated conditions. When the pH was less than 6 or higher than 8, the fluorescence signals detected by immunosensor systems were obviously reduced with the decrease or increase of pH. When the pH ranged from 6 to 8, IC50 and the linear working range of MC-LR calculated from the detection curves were 1.01∼1.04 μg/L and 0.12∼10.5 μg/L, respectively, which was favourable for an MC-LR immunoassay. Low concentrations of Cu2+ rarely affected the detection performance of MC-LR. When the concentration of CuSO4 was higher than 5 mg/L, the fluorescence signal detected by EWAI clearly decreased, and when the concentration of CuSO4 was 10 mg/L, the fluorescence signal detected was reduced by 70%. The influence of Cu2+ on the immunoassay could effectively be compromised when chelating reagent EDTA was added to the pre-reaction mixture

Emerging optical materials in sensing and discovery of bioactive compounds

Optical biosensors are used in numerous applications and analytical fields. Advances in these sensor platforms offer high sensitivity, selectivity, miniaturization, and real-time analysis, among many other advantages. Research into bioactive natural products serves both to protect against potentially dangerous toxic compounds and to promote pharmacological innovation in drug discovery, as these compounds have unique chemical compositions that may be characterized by greater safety and efficacy. However, conventional methods for detecting these biomolecules have drawbacks, as they are time-consuming and expensive. As an alternative, optical biosensors offer a faster, simpler, and less expensive means of detecting various biomolecules of clinical interest. In this review, an overview of recent developments in optical biosensors for the detection and monitoring of aquatic biotoxins to prevent public health risks is first provided. In addition, the advantages and applicability of these biosensors in the field of drug discovery, including high-throughput screening, are discussed. The contribution of the investigated technological advances in the timely and sensitive detection of biotoxins while deciphering the pathways to discover bioactive compounds with great health-promoting prospects is envisaged to meet the increasing demands of healthcare systems.The authors gratefully acknowledge funding from the European Regional Development Fund (ERDF) through COMPETE 2020-POCI and Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

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

Towards Autonomous Microcystin Detection: Investigating Methods for Automation

Due to increased anthropogenic activity, severe eutrophication is occurring in bodies of water around the world. Effects include decreased water quality, decreased value of surrounding land and recreational use (estimated loss in revenue of 0.67 and 3.96 U.S. billion dollars per year), and increased occurrence of toxin producing Harmful Algal Blooms (HABs). Microcystins are cyclic peptides made up of 7 amino acids and 800-1100 Daltons in size. They are one of the most predominantly produced of these toxins, and therefore was the focus of this study. Numerous structural variants of microcystin (referred to as congeners) exist, but microcystin-LR is one of the most common, having a World Health Organization (WHO) recommended limit of 1 µg/L in drinking water. In order to make informed public health decisions on potable and recreational water, an automated in situ instrument for detection of microcystin and its nucleic acids is needed. Very few detection systems have reached the market (i.e. Environmental Sample Processor, McLane Laboratories, USA), but all remain prohibitively costly and complex. Currently, research in many fields is directed towards developing a more cost effective automated in situ detection instrument that can collect and filter environmental samples, extract toxins and nucleic acids, and detect and quantify analytes, genes, and gene transcripts. In this study, a sample preparation method for on-filter collection, filtration, and dual extraction of microcystin and nucleic acids was developed during the summer of 2016 on environmental samples from two bodies of water, Lake Winnebago, WI and Veteran’s Park Lagoon, Milwaukee, WI. Results were compared to a traditional laboratory bead beating method. Results showed that the median extraction ratios (quantified by mass spectrometry) obtained with on-filter method compared to bead beat method (comparative recovery) for microcystin congeners MC-LR, MC-YR, MC-RR, and MC-LA were 43% ± 12%, 34% ± 9%, 46% ± 10% and 44% ± 13%, respectively for Lake Winnebago. The median comparative recovery for MC-LR, MC-YR, and MC-RR was 51% ± 9%, 49% ± 12%, and 53% ± 7%, respectively, for Veteran’s Park Lagoon. Total RNA extraction by the on-filter result showed lower and more inconsistent ratios. Comparative recovery values for the Veteran’s Park Lagoon ranged from 6% to 27% and 5% to 64% for Lake Winnebago. Further quantification with RT-qPCR is needed to evaluate extraction efficiency of the desired gene cluster (mcy). Methods that were evaluated for detection of microcystin included chemical derivatization (fluorescent derivatization) and optical signal amplification (direct and indirect hybridization schemes using DNA aptamers and oligonucleotide probes, nicking enzyme assisted fluorescent signal amplification (NEFSA)). Methods evaluated for detection of nucleic acids included optical signal amplification (direct and indirect hybridization, NEFSA, cascading amplification of nucleic acids (CANA)) and nucleic acid amplification (strand displacement amplification (SDA)). Of the techniques tested, SDA gave non-specific or no amplification, fluorescent derivatization was inconsistent, and all hybridization schemes resulted in non-specific binding. Preliminary results from NEFSA and CANA showed promise, but were inconsistent. Therefore, further optimization of reaction conditions is necessary to conclude if either could be viable options for use in an automated in situ detection system in combination with the on-filter sample preparation and extraction technique

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced

Studies on the immunosuppressive effects and detection of naturally-occuring toxins

Episodes of toxin-producing phytoplankton occur worldwide, causing both animal and human fatalities. Toxicity occurs through consumption of phycotoxins, including azaspiracid, which accumulate in filter-feeding shellfish. Microcystins are hepatotoxins, produced mainly by freshwater cyanobacteria. Aflatoxins are potent, fungal hepatocarcinogens, which occur mainly in food and feed products. The purpose of this research was to examine the cytotoxic and immunosuppressive effects of aflatoxins (B1, B2 and G1), azaspiracid-1 and microcystin-LR in vitro, using the murine macrophage cell line, J774A.1. The results clearly demonstrated that azaspiracid and microcystin had a significant effect on host defence functions, through deregulation of IL-6, IL-10, IL12p40 and TNF-α cytokine expression. Microcystin exposure significantly decreased IL-1β expression. ‘Toll-like’ receptor (TLR2 and CD14) expression was altered following aflatoxin exposure, while apoptotic marker (caspase-1) expression was affected following microcystin exposure. This knowledge should be taken into consideration in the implementation of detection limits, aimed at minimising risks to human health through toxin exposure. Increased awareness of the hazards presented by toxins led to the requirement for recombinant antibodies for these targets, for incorporation into sensitive detection immunoassays. This thesis describes the production of leprine and avian immune libraries for azaspiracid and microcystin, respectively. Attempts were made to isolate azaspiracid-specific antibodies with little success. Phage display was utilised to successfully isolate two single chain antibody fragments (scFvs) to microcystin from the avian library. Error-prone PCR resulted in the isolation of a mutant clone which displayed a 2.3-fold improvement in sensitivity by ELISA, with an LOD of 1.4 ng/mL. The mutant scFv displayed an altered cross-reactivity profile to the microcystin variants tested using Biacore™ inhibition analysis. The recombinant antibodies were successfully applied to the development of fluorescence-based immunoassay formats. The biotinylated mutant scFv was incorporated into a slide-based assay format on a functionalised glass substrate (IC50 ~ 1 µg/L). This assay had the potential to accurately detect microcystin and its variants, below the regulatory limit of 1 µg/L. The application of these highly-sensitive recombinant antibodies into rapid and inexpensive fluorescence detection systems could aid in the development of an early warning system for toxin outbreaks

A Novel Antibody-Dependent MC-LR Detecting Biosensor for Early Warning of Harmful Algal Blooms (HABs)

Microcystins (MCs) are toxins produced by cyanobacteria commonly found in harmful algal blooms (HAB) occurring in many surface waters. Due to their toxicity to humans and other organisms, the World Health Organization (WHO) set a guideline of 1 µg/L for microcystin-leucine-arginine (MC-LR) in drinking water. However, current analytical techniques for the detection of MC-LR such as liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA) are costly, bulky, time-consuming, and mostly conducted in a laboratory, requiring highly trained personnel. Therefore, an analytical method that can be used in the field for rapid determination is essential. In this study, an Anti-MC-LR/MC-LR/Cysteamine coated screen-printed carbon electrode (SPCE) biosensor was newly developed to detect MC-LR, bioelectrochemically, in water. The functionalization of the electrode surface was examined using scanning electron microscopy-energy dispersive X-Ray spectroscopy (SEM-EDX) and X-Ray photoelectron spectroscopy (XPS). The sensor performance was evaluated by electrochemical impedance spectroscopy (EIS), obtaining a linear working range of MC-LR concentrations between 0.1 and 100 µg/L with a limit of detection (LOD) of 0.69 ng/L. Natural water samples experiencing HABs were then collected and analyzed using the developed biosensor and validated using ELISA, demonstrating the excellent performance of the biosensor with a relative standard deviation (RSD) of 0.65%. The interference and selectivity tests showed a minimal error and RSD values against other common MCs and possible coexisting ions found in water, suggesting high selectivity and low sensitivity of the biosensor. The biosensor showed acceptable functionality with a shelf life of up to 12 weeks. Overall, the Anti-MC-LR/MC-LR/Cysteamine/SPCE biosensors can be an innovative solution with characteristics that allow for in situ, low-cost, and easy-to-use capabilities which are essential for developing an overarching and integrated smart environmental management system

The development of microfluidic platforms for environmental analysis

There is currently a gap in the use of centrifugal microfluidics in the field environmental sensing. The purpose of this thesis was to develop new and innovative centrifugal microfluidic platforms, which could enhance current environmental monitoring strategy limitations; portability and in-situ capability, cost-effectiveness, generical design for multi-analyte detectability, and the minimal required end-user interaction. Included in the main body of the thesis will be a review article, providing the theoretical perspectives which have been demonstrated for microfluidic applications in other domains and recommendations for adaptation towards environmental sensing using centrifugal microfluidics, and three novel papers on the staged development of a multi- toxin detection platform aimed to be incorporated within the fully deployable MariaBox (Marine environmental in-situ assessment and monitoring toolBox, co-funded by the European Commission: contract no.614088) system. The aspects covered across these three original articles includes the development of a centrifugal microfluidic platform with complementary fluorescence detection system as an initial test bed for toxin bio- assay integration on-disc, progression of current centrifugally-automatable pneumatic microvalve mechanisms for increased actuation predictability, and the further combination of both of these detection and microvalve mechanisms for a complete on- disc, multi-toxin detection platform which has been designed specifically to be compatible with the deployable MariaBox platform